Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The multi-protective effect of IL-37-Smad3 against ox-LDL induced dysfunction of endothelial cells.

doi: 10.1016/j.biopha.2024.116268

Figure Lengend Snippet: Fig. 3. RH-IL37 (1 ng/mL) treatment suppressed ox-LDL induced endoplasmic reticulum stress expression in RCAECs dependent on Smad3 factor. The protein expression of IRE1-XBP1, PERK-eIF2α-ATF4, ATF6 pathway, and ERS markers CHOP, GRP78 in the cytoplasm of RCAECs assessed following exposed to ox-LDL (80 µg/mL) after pretreatment with or without RH-IL37 (1 ng/mL) or Smad3 silencing. The results demonstrated that ox-LDL increased the expression of IRE1- XBP1, PERK-eIF2α-ATF4, and ATF6 pathway components, as well as CHOP, GRP78 markers in RCAECs,. However, intervention with IL-37 significantly

Article Snippet: Furthermore, the antibodies against XBP1s (rabbit monoclonal antibody #4868–1-AP), ATF4 (rabbit monoclonal antibody #10835–1-AP), ALP (mouse monoclonal antibody #60294–1-Ig), RUNX2 (Rabbit monoclonal antibody #20700–1-AP), and BMP-2 (mouse monoclonal antibody #66383–1-Ig) were obtained from Proteintech (Wuhan Sanying, Wuhan, Hubei, P.R.China).

Techniques: Expressing

Journal: Nature communications

Article Title: Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.

doi: 10.1038/ncomms7239

Figure Lengend Snippet: Figure 6 | PERK-eIF2a-ATF4 axis regulates Yap transcription during the UPR. (a) Western blot analysis of Hippo signalling elements in HepG2 cells treated with 1 mg ml 1 TM for different lengths of time. Western blotting (b) or quantitative PCR (qPCR) (c) analysis of Yap expression in PERK-knockdown HepG2 cells treated with 1 mg ml 1 TM for 8 h. (d) Extracted data from a previously reported microarray analysis52 of UPR elements, Yap and CTGF levels in the livers of WT and Perkfl/fl-AlbCre (Perk / ) mice followed by a 6-h 1 mg kg 1 TM treatment. (e) qPCR analysis of Yap expression in HepG2 cells overexpressing ATF4. (f) Chromatin immunoprecipitation (ChIP) assay showing that ATF4 bound to the Yap promoter in HepG2 cells followed by an 8-h 1 mg ml 1 TM treatment. (g) Luciferase assay showing that ATF4 enhanced Yap-1000-WT but not Yap-1000-DATF4 promoter activity levels. (h) Western blotting showing that ATF4 regulated Yap expression. (i) Western blotting showing that eIF2a but not a mutated eIF2a (S51A) increased Yap protein levels in HepG2 cells. Knock-in of the eIF2a (S51A) gene led to significantly reduced liver masses and liver/body weight ratios in the Mst1/2-deficient (DKO) mice (j) and decreased the protein levels of ATF4 and Yap in DKO livers (k), n ¼ 5. Data were assessed by Student’s t-test and represented as mean±s.d. ns, not significant, *Po0.05, **Po0.01, ***Po0.001 compared with respective controls or as indicated. Data are representative of at least three independent experiments.

Article Snippet: One per cent of input was removed and the lysates were immunoprecipitated with 2 mg of rabbit anti-ATF4 antibody (Proteintech Group Inc.), species-matched IgG control antibody or 40ml of A/G-Sepharose beads for 2 h. Salmon sperm DNA/protein A/GSepharose beads were added to the polyclonal ATF4 and IgG control immunoprecipitations for incubation overnight.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Microarray, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Knock-In

Journal: Nature communications

Article Title: Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.

doi: 10.1038/ncomms7239

Figure Lengend Snippet: Figure 9 | Elevated PERK-eIF2a-Yap signalling is associated with the development of mouse and human HCC. (a–c) A low dose of TM promotes cell hyperproliferation. Western blot analysis of PERK signalling and Yap levels in HepG2 cells treated with a low dose of TM (10 ng ml 1) for the indicated number of days (a). Low-dose TM-treated HepG2 cells exhibited the formation of significantly more colonies (b) and formed tumours of larger masses in the nude mice (c) compared with the dimethylsulfoxide (DMSO)-treated control cells, n ¼ 8. The black arrow indicates the TM-treated tumour cells; the white arrow denotes the control cells. Data were assessed by Student’s t test and represented as mean±s.d. ***Po0.001. (d) Western blotting with the indicated antibodies in liver tissues isolated from DEN- or DEN-plus-TUDCA-treated mice. (e,f) TUDCA treatment reduced liver masses (e) and the numbers of HCC tumors (f) in DEN-challenged mice, n ¼ 8. Data assessment is same as in c. ***Po0.001. (g) Yapfl/ þ-AblCre mice exhibited significantly reduced numbers of DEN-induced HCC tumours compared with control WT mice. Data assessment is same as in c. ***Po0.001. (h,i) Western blot analysis of Bip, PERK, phospo-eIF2a and Yap in liver cancer tissue (T) and non-tumorous liver tissue (N) isolated from one patient. A total of six representative paired samples are shown (h). See Supplementary Fig. 11 for the remaining 60 paired samples. The intensities of the immunoblot bands were quantified using the Imagine gel software. The ratio of the relative expression of the indicated proteins in the T and N from one patient was plotted and applied with the linear regression t-test (i). (j) A proposed working model for the interplay of the Hippo pathway and the UPR signalling for cell fate determination. Under ER stress conditions, the PERK-mediated eIF2a phosphorylation increases ATF4 expression for the subsequent induction of Yap. Activated Yap increases UPR activity, the size and Ca2 þ storage capacity of ER for cell survival. When ER stress is excessive and homeostasis is not restored, Hippo signalling is activated for stabilizing GADD34/CReP proteins, which assemble with PP1 to form a complex to dephosphorylate eIF2a and reduces the PP1 abundance in the nucleus. In the end, increased Yap phosphorylation inhibits UPR activity and ER membrane expansion and eventually promotes cell death.

Article Snippet: One per cent of input was removed and the lysates were immunoprecipitated with 2 mg of rabbit anti-ATF4 antibody (Proteintech Group Inc.), species-matched IgG control antibody or 40ml of A/G-Sepharose beads for 2 h. Salmon sperm DNA/protein A/GSepharose beads were added to the polyclonal ATF4 and IgG control immunoprecipitations for incubation overnight.

Techniques: Western Blot, Control, Isolation, Software, Expressing, Phospho-proteomics, Activity Assay, Membrane